Project Summary/Abstract Cell migration is required for many important physiological and pathological processes such as embryonic development, wound healing, and cancerous invasion. As a process that involves concerted action of multiple ensembles of molecules over the length of the entire cell, cell migration cannot be understood using conventional molecular approaches alone without considering sensing, actuation, and control at the whole cell level. This project seeks to approach migrating cells in a top-down manner as an integrated mechanochemical system. Based on observations that likely represent the manifestation of a complex network of molecular interactions, we may deduct how the underlying machine operates. The project will be facilitated by the development of new technologies, including 3D printing of polyacrylamide hydrogels and machine learning for cell tracking, traction force microscopy, and super resolution imaging. We will address three important aspects. First, we will ask how cells initiate migration through a process known as symmetry breaking, which causes a symmetrically spreading cell to initiate directional migration. We will examine various anisotropic properties of the substrate as potential symmetry breaking cues. In addition, the function of filopodia as possible sensors for symmetry breaking will be studied with imaging and pharmacological approaches. Second, we will address several poorly understood aspects of 2D and 3D cell migration. By following migrating cells over a long distance at a high magnification, we expect to place the newly discovered process of contact following in the context of cell collectives. To understand how cell shape control, cell-cell interaction, and cell migration respond to 3D environment, we will use 3D printed polyacrylamide to create model systems and systematically vary geometrical and mechanical parameters. We will then extend the experiments to decellularized lung scaffolds, which have been used for tissue engineering, to determine how migration characteristics in 3D is related to the promotion of tissue formation. Another overlooked area we will examine is the function of the tail in defining cell polarity and mediating contact following. Third, we will seek mechanistic understanding of cellular responses to cyclic stretching, which occurs in various tissues. A novel imaging approach will allow us to determine the responses during the stretching and relaxation phase respectively. A combination of experimentation and computer modeling is planned to explain why epithelial cells respond to static stretching along the direction of forces but perpendicularly in response to cyclic stretching. We will also test the hypothesis that responses to cyclic stretching can cause cell intercalation, a fundamentally important process in embryonic morphogenesis. We expect our results to complement studies at the molecular level and bring paradigm shifting insights into cell migration for both basic cell biology and repair of tissue functions.